We are continuing our efforts in characterizing the lens and processes that may occur in cataract development. There are three aims in our research efforts: determination of the sequences of human crystallins, development of the in vitro lens incubation system, and examination of stress on lenses and lens crystallins. Each of these areas complements the other and builds a base on which to study how the lens resists cataract development and the stresses that will eventually lead of opacification. The first area of work has been the determination of the sequence of human beta B2-crystallin and human gammaS-crystallin. The human beta B2- crystallin is the principal beta crystallin in the lens. Two human lens complementary deoxyribonucleic acid (cDNA) libraries were made. One of the libraries was to adult lens and one was to fetal lens. The beta B2-crystal n cDNA was cloned and sequenced from these libraries and the deduced amino acid sequence was determined. Work also continued on the promoter region of this crystallin in order to develop a construct that would be developmentally regulated in transgenic animals. ~S-crystallin is a protei that increases in content in the lens with age. The amount of this crystallin is very often decreased with the advent of human cataract formation. The ~S-crystallin sequence was determined by a combination of cDNA sequencing, electrospray ionization mass spectrometry, and fast atom bombardment mass spectrometry. In vitro lens incubation is currently one of the only ways to study the effect of environmental stresses on whole lens metabolism. Work has progressed to establish criteria for determining which of the in vitro incubated lenses has metabolic integrity and has not been injured in the dissection process. A simple test has been developed to determine the integrity of the lens in vitro in as few as 30 minutes after the start of t incubation procedure. Studies have been done in vitro to determine the response of the whole lens to oxidative insult using the content and mRNA levels of catalase, an enzyme responsible for protection against H2O2. Glutathione levels of stressed lenses have also been measured to determine the reason for the loss of this vital constituent. The effects of environmental stresses have also been studied using cell culture systems. The cell line used in these studies constitutively expres s `B-crystallin. Effects of oxidative stress and heat shock have been investigated on this crystallin that is thought to serve as a molecular chaperon.